LOCUS       sequence-174750-        6076 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET LIC cloning vector for Biotin-His6-tev-yORF-mRuby2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6076)
  TITLE     C-terminal fluorescent fusion vectors for E. coli expression
REFERENCE   2  (bases 1 to 6076)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pET mRuby2 LIC cloning vector (H6-mRuby2)
FEATURES             Location/Qualifiers
     source          1..6076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     818..837
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(857..934)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1140..1159
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1243..1262
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1243..1261
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1262..1286
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1301..1323
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1336..1380
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1381..1398
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1399..1419
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1456..2163
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     CDS             2182..2199
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2252..2270)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2266..2313
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2350..2805
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2437..2456)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2647..2668
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2898..3713)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2898..3713)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3621..3640
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3705..3724
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3835..4423
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4324..4343
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4577..4594
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4609..4748
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4674..4693)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4834..4856
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4850..5041)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5850..6076,1..856))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
       61 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
      121 tggcaaccag catcgcagtg ggaacgatac cctcattcag catttgcatg gtttgttgaa
      181 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
      241 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
      301 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
      361 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
      421 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
      481 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
      541 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
      601 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
      661 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
      721 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
      781 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
      841 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
      901 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
      961 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     1021 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     1081 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     1141 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     1201 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
     1261 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     1321 agatatacca tgggaggcct gaacgatatt tttgaagcgc agaaaattga atggcatgaa
     1381 caccatcacc atcaccatga aaacctgtac ttccaatcca atattggtag tgggagcaac
     1441 ggcagcagcg gatccgtgtc taagggcgaa gagctgatca aggaaaatat gcgtatgaag
     1501 gtggtcatgg aaggttcggt caacggccac caattcaaat gcacaggtga aggagaaggc
     1561 aatccgtaca tgggaactca aaccatgagg atcaaagtca tcgagggagg acccctgcca
     1621 tttgcctttg acattcttgc cacgtcgttc atgtatggca gccgtacttt tatcaagtac
     1681 ccgaaaggca ttcctgattt ctttaaacag tcctttcctg agggttttac ttgggaaaga
     1741 gttacgagat acgaagatgg tggagtcgtc accgtcatgc aggacaccag ccttgaggat
     1801 ggctgtctcg tttaccacgt ccaagtcaga ggggtaaact ttccctccaa tggtcccgtg
     1861 atgcagaaga agaccaaggg ttgggagcct aatacagaga tgatgtatcc agcagatggt
     1921 ggtctgaggg gatacactca tatggcactg aaagttgatg gtggtggcca tctgtcttgc
     1981 tctttcgtaa caacttacag gtcaaaaaag accgtcggga acatcaagat gcccggtatc
     2041 catgccgttg atcaccgcct ggaaaggtta gaggaaagtg acaatgaaat gttcgtagta
     2101 caacgcgaac acgcagttgc caagttcgcc gggcttggtg gtgggatgga cgagctgtac
     2161 aagtaagcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     2221 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2281 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     2341 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2401 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2461 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2521 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2581 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2641 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2701 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2761 aaaaatttaa cgcgaatttt aacaaactag taacgtttac aatttcaggt ggcacttttc
     2821 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2881 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     2941 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     3001 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     3061 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     3121 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     3181 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     3241 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     3301 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatgttt
     3361 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     3421 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     3481 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     3541 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     3601 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     3661 ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc
     3721 cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg
     3781 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3841 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3901 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3961 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     4021 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4081 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4141 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4201 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4261 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4321 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4381 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4441 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4501 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4561 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4621 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     4681 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     4741 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4801 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4861 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     4921 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     4981 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     5041 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     5101 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     5161 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     5221 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     5281 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     5341 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     5401 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     5461 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc
     5521 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     5581 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     5641 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     5701 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     5761 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     5821 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5881 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5941 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     6001 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     6061 tgtttgatgg tggtta
//
